Publications by authors named "Banabihari Giri"

18 Publications

  • Page 1 of 1

Emerging urinary alpha-synuclein and miRNA biomarkers in Parkinson's disease.

Metab Brain Dis 2021 Apr 21. Epub 2021 Apr 21.

Department of Physical Therapy, College of Allied Health Sciences, Augusta University, 987 St. Sebastian Way, Augusta, GA, 30912, USA.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases after Alzheimer's disease (AD), afflicting adults above the age of sixty irrespective of gender, race, ethnicity, and social status. PD is characterized by motor dysfunctions, displaying resting tremor, rigidity, bradykinesia, and postural imbalance. Non-motor symptoms, including rapid eye movement (REM) behavior disorder, constipation, and loss of sense of smell, typically occur many years before the appearance of the PD motor symptoms that lead to a diagnosis. The loss of dopaminergic neurons in the substantia nigra, which leads to the motor symptoms seen in PD, is associated with the deposition of aggregated, misfolded α-Synuclein (α-Syn, SNCA) proteins forming Lewy Bodies. Additionally, dysregulation of miRNA (a short form of mRNA) may contribute to the developing pathophysiology in PD and other diseases such as cancer. Overexpression of α-Syn and miRNA in human samples has been found in PD, AD, and dementia. Therefore, evaluating these molecules in urine, present either in the free form or in association with extracellular vesicles of biological fluids, may lead to early biomarkers for clinical diagnosis. Collection of urine is non-invasive and thus beneficial, particularly in geriatric populations, for biomarker analysis. Considering the expression and function of α-Syn and miRNA, we predict that they can be used as early biomarkers in the diagnosis and prognosis of neurodegenerative diseases.
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http://dx.doi.org/10.1007/s11011-021-00735-2DOI Listing
April 2021

Niacin Ameliorates Neuro-Inflammation in Parkinson's Disease via GPR109A.

Int J Mol Sci 2019 Sep 14;20(18). Epub 2019 Sep 14.

Charlie Norwood VA Medical Center, Augusta, GA 30904, USA.

In this study, we used macrophage RAW264.7 cells to elucidate the molecular mechanism underlying the anti-inflammatory actions of niacin. Anti-inflammatory actions of niacin and a possible role of its receptor GPR109A have been studied previously. However, the precise molecular mechanism of niacin's action in reducing inflammation through GPR109A is unknown. Here we observed that niacin reduced the translocation of phosphorylated nuclear kappa B (p-NF-κB) induced by lipopolysaccharide (LPS) in the nucleus of RAW264.7 cells. The reduction in the nuclear translocation in turn decreased the expression of pro-inflammatory cytokines IL-1β, IL-6 in RAW264.7 cells. We observed a decrease in the nuclear translocation of p-NF-κB and the expression of inflammatory cytokines after knockdown of GPR109A in RAW264.7 cells. Our results suggest that these molecular actions of niacin are mediated via its receptor GPR109A (also known as HCAR2) by controlling the translocation of p-NF-κB to the nucleus. Overall, our findings suggest that niacin treatment may have potential in reducing inflammation by targeting GPR109A.
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http://dx.doi.org/10.3390/ijms20184559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770365PMC
September 2019

Niacin modulates macrophage polarization in Parkinson's disease.

J Neuroimmunol 2018 07 4;320:76-79. Epub 2018 May 4.

Department of Oral Biology, Dental College of Georgia, Augusta University, Augusta, GA, USA; Department of Neurology, Medical College of Georgia, Augusta University, Augusta, GA, USA. Electronic address:

Neuroinflammation remains a central piece in Parkinson's disease (PD) pathophysiology. However, mechanisms by which PD links to the neuroinflammation remain elusive. Here, for the first time, we report that lower dose of niacin in PD patients may affect macrophage polarization from M1 (pro-inflammatory) to M2 (counter-inflammatory) profile through the niacin receptor GPR109A. Skew in the peripheral macrophages were accompanied by improved quality of life assessments in patients. Low dose niacin supplementation may be beneficial in PD, boosting anti-inflammatory processes and suppressing inflammation. Varied niacin dosages for longer durations may further reveal the potential role of anti-inflammatory interventions in PD progression.
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http://dx.doi.org/10.1016/j.jneuroim.2018.05.002DOI Listing
July 2018

expression in acute myeloid leukemia.

Oncotarget 2018 Jan 15;9(7):7442-7452. Epub 2017 Dec 15.

Present address: Stony Brook University Cancer Center, Stony Brook, NY, USA.

Similar signaling pathways could operate in both normal hematopoietic stem and progenitor cells (HSPCs) and leukemia stem cells (LSCs). Thus, targeting LSCs signaling without substantial toxicities to normal HSPCs remains challenging. SALL1, is a member of the transcriptional network that regulates stem cell pluripotency, and lacks significant expression in most adult tissues, including normal bone marrow (NBM). We examined the expression and functional characterization of SALL1 in NBM and in acute myeloid leukemia (AML) using and assays. We showed that SALL1 is expressed preferentially in LSCs- enriched CD34+CD38- cell subpopulation but not in NBM. SALL1 inhibition resulted in decreased cellular proliferation and in inferior AML engraftment in NSG mice and it was also associated with upregulation of PTEN and downregulation of m-TOR, β-catenin, and NF-қB expression. These findings suggest that SALL1 inhibition interrupts leukemogenesis. Further studies to validate SALL1 as a potential biomarker for minimal residual disease (MRD) and to determine SALL1's role in prognostication are ongoing. Additionally, pre-clinical evaluation of SALL1 as a therapeutic target in AML is warranted.
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http://dx.doi.org/10.18632/oncotarget.23448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800914PMC
January 2018

Mutational Dissection of Telomeric DNA Binding Requirements of G4 Resolvase 1 Shows that G4-Structure and Certain 3'-Tail Sequences Are Sufficient for Tight and Complete Binding.

PLoS One 2015 14;10(7):e0132668. Epub 2015 Jul 14.

Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina, 27157, United States of America.

Ends of human chromosomes consist of the six nucleotide repeat d[pTTAGGG]n known as telomeric DNA, which protects chromosomes. We have previously shown that the DHX36 gene product, G4 Resolvase 1 (G4R1), binds parallel G-quadruplex (G4) DNA with an unusually tight apparent Kd. Recent work associates G4R1 with the telomerase holoenzyme, which may allow it to access telomeric G4-DNA. Here we show that G4R1 can tightly bind telomeric G4-DNA, and in the context of the telomeric sequence, we determine length, sequence, and structural requirements sufficient for tight G4R1 telomeric binding. Specifically, G4R1 binds telomeric DNA in the K+-induced "3+1" G4-topology with an apparent Kd = 10 ± 1.9 pM, a value similar as previously found for binding to unimolecular parallel G4-DNA. G4R1 binds to the Na+-induced "2+2" basket G4-structure formed by the same DNA sequence with an apparent Kd = 71 ± 2.2 pM. While the minimal G4-structure is not sufficient for G4R1 binding, a 5' G4-structure with a 3' unstructured tail containing a guanine flanked by adenine(s) is sufficient for maximal binding. Mutations directed to disrupt G4-structure similarly disrupt G4R1 binding; secondary mutations that restore G4-structure also restore G4R1 binding. We present a model showing that a replication fork disrupting a T-loop could create a 5' quadruplex with an opened 3'tail structure that is recognized by G4R1.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0132668PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4501837PMC
May 2016

Probing the role of Proline in the antimicrobial activity and lipopolysaccharide binding of indolicidin.

J Colloid Interface Sci 2015 Aug 25;452:148-159. Epub 2015 Apr 25.

Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India. Electronic address:

Hypothesis: Indolicidin (ILPWKWPWWPWRR-NH2), an antimicrobial peptide from bovine neutrophils, possesses significant antibacterial activity. An interesting feature of indolicidin is its unusually high content of Tryptophan and Proline residues. While the involvement of Tryptophan has been studied for its hemolytic and antibacterial activity, little is known about the roles played by Proline in these aspects. We herein investigate the structure and biological activities of indolicidin, where Proline at either one or more of the 3rd, 7th, 10th positions has been replaced by Alanine to better understand its structure and biological function.

Experiments: Structural aspects of Proline residues of indolicidin and its effect on antimicrobial activity were elucidated by replacing Proline residues with Alanine. Minimum inhibitory concentration (MIC) and scanning electron microscopy (SEM) experiments provide substantial evidence for the importance of Proline residues for antimicrobial activity and cell wall disintegration. Binding affinity of the peptides to Lipopolysaccharide (LPS) was investigated using fluorescence spectroscopy and dynamic light scattering (DLS) in conjunction with (31)PNMR spectroscopy and confirmed the disintegration of LPS layer.

Findings: Our study reveals that Proline residues are necessary for interaction of indolicidin with LPS and establishes the significance of the third and tenth Proline residues for its antimicrobial activity. We believe that the presence of so many Proline residues provides the molecule a selective advantage of adopting different conformations varying from a globular, closed conformation to an open extended conformation, and even to a wedge-shaped conformation, which account for the diverse mechanisms of action of indolicidin.
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http://dx.doi.org/10.1016/j.jcis.2015.04.031DOI Listing
August 2015

A silver cluster-DNA equilibrium.

Anal Chem 2013 Oct 26;85(20):9868-76. Epub 2013 Sep 26.

Department of Chemistry, Furman University , Greenville, South Carolina 29613, United States.

DNA encapsulates silver clusters, and these hybrid nanomaterials form molecular sensors. We discuss a silver cluster-oligonucleotide sensor with four characteristics. First, a specific reporting cluster forms within a single-stranded DNA. This template uses the 5' cluster domain CCCCAACTCCTT with different 3' recognition sites for complementary oligonucleotides. The modular composite strand exclusively forms a cluster with λmax = 400 nm and with low emission. Conjugates were chromatographically purified, and their elemental analysis measured a cluster adduct with ∼11 silver atoms. Second, hybridization transforms the cluster. Size exclusion chromatography shows that the 3' recognition sites of the single-stranded conjugates hybridize with their complements. This secondary structural change both shifts cluster absorption from 400 to 490 nm and develops emission at 550 nm. Third, cluster size remains intact. Like their violet predecessors, purified blue-green clusters have ∼11 silver atoms. Cluster integrity is further supported by extracting the complement from the blue-green conjugate and reversing the spectral changes. Fourth, the cluster transformation is an equilibrium. Complementary strands generate an isosbestic point and thus directly link single-stranded hosts for the violet cluster and their hybridized analogs for the blue-green cluster. This equilibrium shifts with temperature. A van't Hoff analysis shows that longer and more stable duplexes favor the blue-green cluster. However, hybridized cluster hosts are less stable than their native DNA counterparts, and stability further degrades when short complements expose nucleobases within S1-S2. Duplex instability suggests that unpaired nucleobases coordinate the violet cluster and favor the single-stranded sensor. A balance between innate hybridization and exogenous folding highlights a distinct feature of silver clusters for sensing: they are both chromophoric reporters and ligands that modulate analyte-sensor interactions.
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http://dx.doi.org/10.1021/ac4028559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4532306PMC
October 2013

Silver clusters as both chromophoric reporters and DNA ligands.

Anal Chem 2013 Feb 1;85(4):2183-90. Epub 2013 Feb 1.

Department of Chemistry, Furman University, Greenville, South Carolina 29613, United States.

Molecular silver clusters conjugated with DNA act as analyte sensors. Our studies evaluate a type of cluster-laden DNA strand whose structure and silver stoichiometry change with hybridization. The sensor strand integrates two functions: the 3' region binds target DNA strands through base recognition while the 5' sequence C(3)AC(3)AC(3)TC(3)A favors formation of a near-infrared absorbing and emitting cluster. This precursor form exclusively harbors an ∼11 silver atom cluster that absorbs at 400 nm and that condenses its single-stranded host. The 3' recognition site associates with a complementary target strand, thereby effecting a 330 nm red-shift in cluster absorption and a background-limited recovery of cluster emission at 790 nm. One factor underlying these changes is sensor unfolding and aggregation. Variations in salt and oligonucleotide concentrations control cluster development by influencing DNA association. Structural studies using fluorescence anisotropy, fluorescence correlation spectroscopy, and size exclusion chromatography show that the sensor-cluster conjugate opens and subsequently dimerizes with hybridization. A second factor contributing to the spectral and photophysical changes is cluster transformation. Empirical silver stoichiometries are preserved through hybridization, so hybridized, dimeric near-infrared conjugates host twice the amount of silver in relation to their violet absorbing predecessors. These DNA structure and net silver stoichiometry alterations provide insight into how DNA-silver conjugates recognize analytes.
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http://dx.doi.org/10.1021/ac303531yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4532293PMC
February 2013

Yin Yang 1 contains G-quadruplex structures in its promoter and 5'-UTR and its expression is modulated by G4 resolvase 1.

Nucleic Acids Res 2012 Feb 12;40(3):1033-49. Epub 2011 Oct 12.

Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.

Yin Yang 1 (YY1) is a multifunctional protein with regulatory potential in tumorigenesis. Ample studies demonstrated the activities of YY1 in regulating gene expression and mediating differential protein modifications. However, the mechanisms underlying YY1 gene expression are relatively understudied. G-quadruplexes (G4s) are four-stranded structures or motifs formed by guanine-rich DNA or RNA domains. The presence of G4 structures in a gene promoter or the 5'-UTR of its mRNA can markedly affect its expression. In this report, we provide strong evidence showing the presence of G4 structures in the promoter and the 5'-UTR of YY1. In reporter assays, mutations in these G4 structure forming sequences increased the expression of Gaussia luciferase (Gluc) downstream of either YY1 promoter or 5'-UTR. We also discovered that G4 Resolvase 1 (G4R1) enhanced the Gluc expression mediated by the YY1 promoter, but not the YY1 5'-UTR. Consistently, G4R1 binds the G4 motif of the YY1 promoter in vitro and ectopically expressed G4R1 increased endogenous YY1 levels. In addition, the analysis of a gene array data consisting of the breast cancer samples of 258 patients also indicates a significant, positive correlation between G4R1 and YY1 expression.
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http://dx.doi.org/10.1093/nar/gkr849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3273823PMC
February 2012

G4 resolvase 1 tightly binds and unwinds unimolecular G4-DNA.

Nucleic Acids Res 2011 Sep 17;39(16):7161-78. Epub 2011 May 17.

Department of Cancer Biology and the Comprehensive Cancer Center of Wake Forest University School of Medicine, Winston-Salem, USA.

It has been previously shown that the DHX36 gene product, G4R1/RHAU, tightly binds tetramolecular G4-DNA with high affinity and resolves these structures into single strands. Here, we test the ability of G4R1/RHAU to bind and unwind unimolecular G4-DNA. Gel mobility shift assays were used to measure the binding affinity of G4R1/RHAU for unimolecular G4-DNA-formed sequences from the Zic1 gene and the c-Myc promoter. Extremely tight binding produced apparent K(d)'s of 6, 3 and 4 pM for two Zic1 G4-DNAs and a c-Myc G4-DNA, respectively. The low enzyme concentrations required for measuring these K(d)'s limit the precision of their determination to upper boundary estimates. Similar tight binding was not observed in control non-G4 forming DNA sequences or in single-stranded DNA having guanine-rich runs capable of forming tetramolecular G4-DNA. Using a peptide nucleic acid (PNA) trap assay, we show that G4R1/RHAU catalyzes unwinding of unimolecular Zic1 G4-DNA into an unstructured state capable of hybridizing to a complementary PNA. Binding was independent of adenosine triphosphate (ATP), but the PNA trap assay showed that unwinding of G4-DNA was ATP dependent. Competition studies indicated that unimolecular Zic1 and c-Myc G4-DNA structures inhibit G4R1/RHAU-catalyzed resolution of tetramolecular G4-DNA. This report provides evidence that G4R1/RHAU tightly binds and unwinds unimolecular G4-DNA structures.
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http://dx.doi.org/10.1093/nar/gkr234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3167620PMC
September 2011

Role of the amino terminal RHAU-specific motif in the recognition and resolution of guanine quadruplex-RNA by the DEAH-box RNA helicase RHAU.

Nucleic Acids Res 2010 Oct 14;38(18):6219-33. Epub 2010 May 14.

Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, Maulbeerstrasse 66, 4058 Basel, Switzerland.

Under physiological conditions, guanine-rich sequences of DNA and RNA can adopt stable and atypical four-stranded helical structures called G-quadruplexes (G4). Such G4 structures have been shown to occur in vivo and to play a role in various processes such as transcription, translation and telomere maintenance. Owing to their high-thermodynamic stability, resolution of G4 structures in vivo requires specialized enzymes. RHAU is a human RNA helicase of the DEAH-box family that exhibits a unique ATP-dependent G4-resolvase activity with a high affinity and specificity for its substrate in vitro. How RHAU recognizes G4-RNAs has not yet been established. Here, we show that the amino-terminal region of RHAU is essential for RHAU to bind G4 structures and further identify within this region the evolutionary conserved RSM (RHAU-specific motif) domain as a major affinity and specificity determinant. G4-resolvase activity and strict RSM dependency are also observed with CG9323, the Drosophila orthologue of RHAU, in the amino terminal region of which the RSM is the only conserved motif. Thus, these results reveal a novel motif in RHAU protein that plays an important role in recognizing and resolving G4-RNA structures, properties unique to RHAU among many known RNA helicases.
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http://dx.doi.org/10.1093/nar/gkq372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2952847PMC
October 2010

Fat-storing multilocular cells expressing CCR5 increase in the thymus with advancing age: potential role for CCR5 ligands on the differentiation and migration of preadipocytes.

Int J Med Sci 2009 Dec 4;7(1):1-14. Epub 2009 Dec 4.

Laboratories of Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.

Age-associated thymic involution is characterized by decreased thymopoiesis, adipocyte deposition and changes in the expression of various thymic microenvironmental factors. In this work, we characterized the distribution of fat-storing cells within the aging thymus. We found an increase of unilocular adipocytes, ERTR7(+) and CCR5(+ )fat-storing multilocular cells in the thymic septa and parenchymal regions, thus suggesting that mesenchymal cells could be immigrating and differentiating in the aging thymus. We verified that the expression of CCR5 and its ligands, CCL3, CCL4 and CCL5, were increased in the thymus with age. Hypothesizing that the increased expression of chemokines and the CCR5 receptor may play a role in adipocyte recruitment and/or differentiation within the aging thymus, we examined the potential role for CCR5 signaling on adipocyte physiology using 3T3-L1 pre-adipocyte cell line. Increased expression of the adipocyte differentiation markers, PPARgamma2 and aP2 in 3T3-L1 cells was observed under treatment with CCR5 ligands. Moreover, 3T3-L1 cells demonstrated an ability to migrate in vitro in response to CCR5 ligands. We believe that the increased presence of fat-storing cells expressing CCR5 within the aging thymus strongly suggests that these cells may be an active component of the thymic stromal cell compartment in the physiology of thymic aging. Moreover, we found that adipocyte differentiation appear to be influenced by the proinflammatory chemokines, CCL3, CCL4 and CCL5.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2792732PMC
http://dx.doi.org/10.7150/ijms.7.1DOI Listing
December 2009

Reduction of T cell-derived ghrelin enhances proinflammatory cytokine expression: implications for age-associated increases in inflammation.

Blood 2009 May 26;113(21):5202-5. Epub 2009 Mar 26.

Laboratory of Immunology, National Institute on Aging-Intramural Research Program, National Institutes of Health, Baltimore, MD 21224, USA.

Ghrelin (Grln) is a peptide hormone that is predominantly produced in the stomach and stimulates appetite and induces growth hormone (GH) release. We have previously reported that ghrelin is also expressed in T cells and exerts prothymic and anti-inflammatory effects. However, the biologic relevance of T cell-derived ghrelin remains to be determined. Here, we report that acylated-bioactive ghrelin is expressed in human T cells and preferentially segregates within the lipid raft domains upon TCR ligation. The RNA interference (RNAi)-mediated down-regulation of ghrelin in primary human T cells activates IkB, and increases Th1 cytokines and IL-17 secretion. Ghrelin expression declines with increasing age in spleen and T cells and exogenous ghrelin administration in old mice reduces proinflammatory cytokines. These findings demonstrate that ghrelin functions in an autocrine and paracrine capacity to regulate proinflammatory cytokine expression in human and murine T cells and may contribute in regulating "inflamm-aging."
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http://dx.doi.org/10.1182/blood-2008-09-181255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2686189PMC
May 2009

Induction of 150-kDa adenosine deaminase that acts on RNA (ADAR)-1 gene expression in normal T lymphocytes by anti-CD3-epsilon and anti-CD28.

Immunology 2007 Dec 25;122(4):623-33. Epub 2007 Sep 25.

Section on Rheumatology and Immunology, Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.

We and other investigators have demonstrated up-regulation of the expression of the RNA-editing gene 150-kDa adenosine deaminase that acts on RNA (ADAR1) in systemic lupus erythematosus (SLE) T cells and B cells, peripheral blood mononuclear cells (PBMC), natural killer (NK) cells. The presence of a small proportion of activated T cells is the hallmark of SLE. Therefore, it was hypothesized that 150-kDa ADAR1 gene expression is induced by the physiological activation of T cells. To examine this hypothesis, normal T cells were activated by anti-CD3-epsilon plus anti-CD28 for various time periods from 0 to 48 hr. The expression of 110-kDa and 150-kDa ADAR1, and interleukin (IL)-2 and beta-actin gene transcripts was analysed. An approximately fourfold increase in 150-kDa ADAR1 gene expression was observed in activated T cells. ADAR2 gene transcripts are substrates for ADAR1 and ADAR2 enzymes. Therefore, we assessed the role of the 150-kDa ADAR enzyme in editing of ADAR2 gene transcripts. In activated T cells, site-selective editing of the -2 site was observed. Previous studies indicate that this site is predominantly edited by ADAR1. In addition to this, novel editing sites at base positions -56, -48, -45, -28, -19, -15, +46 and +69 were identified in activated T cells. On the basis of these results, it is proposed that 150-kDa ADAR1 gene expression is selectively induced in T cells by anti-CD3-epsilon and anti-CD28 stimulation and that it may play a role in site-selective editing of gene transcripts and in altering the functions of several gene products of T cells during activation and proliferation.
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http://dx.doi.org/10.1111/j.1365-2567.2007.02709.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2266038PMC
December 2007

CXCL12-induced partitioning of flotillin-1 with lipid rafts plays a role in CXCR4 function.

Eur J Immunol 2007 Aug;37(8):2104-16

Laboratory of Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.

Lipid rafts play an important role in signal integration and in the cellular activation of a number of cytokine and growth factor receptors. It has recently been demonstrated that flotillin proteins are recruited to lipid raft microdomains upon cellular activation and play a role in neural cell regeneration, receptor signaling and lymphocyte activation. However, little is known about the relevance of the flotillin proteins during T cell responses to chemoattractant stimulation. To this end, cytoplasmic and lipid raft fractions from human T cells were analyzed for flotillin protein redistribution prior to and after CXCL12 stimulation. Flotillin-1, but not flotillin-2, redistributes to lipid rafts upon CXCR4 ligation. Moreover, in CXCL12-treated T cells, flotillin-1 also associates with several raft proteins including LAT, CD48 and CD11a but not Lck. In addition, an increase in CXCR4 association with flotillin-1 in lipid rafts was observed after chemokine treatment. RNAi technology was also utilized to inhibit the expression of flotillin-1, resulting in an inhibition of CXCL12-mediated signaling, function and CXCR4 recruitment into lipid rafts. Together, these data suggest that the increased association of cellular flotillin-1 with lipid raft microdomains during chemokine exposure may play an important role in chemokine receptor signaling and receptor partitioning with lipid rafts.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2271046PMC
http://dx.doi.org/10.1002/eji.200636680DOI Listing
August 2007

Dynamic reorganization of chemokine receptors, cholesterol, lipid rafts, and adhesion molecules to sites of CD4 engagement.

Exp Cell Res 2005 Apr 23;304(2):559-69. Epub 2004 Dec 23.

Laboratory of Immunology, National Institute on Aging, Intramural Research Program, NIH, DHHS, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA.

T cell polarization and redistribution of cellular components are critical to processes such as activation, migration, and potentially HIV infection. Here, we investigate the effects of CD4 engagement on the redistribution and localization of chemokine receptors, CXCR4 and CCR5, adhesion molecules, and lipid raft components including cholesterol, GM1, and glycosyl-phosphatidylinositol (GPI)-anchored proteins. We demonstrate that anti-CD4-coated beads (alpha CD4-B) rapidly induce co-capping of chemokine receptors as well as GPI-anchored proteins and adhesion molecules with membrane cholesterol and lipid rafts on human T cell lines and primary T cells to the area of bead-cell contact. This process was dependent on the presence of cellular cholesterol, cytoskeletal reorganization, and lck signaling. Lck-deficient JCaM 1.6 cells failed to cap CXCR4 or lipid rafts to alpha CD4-B. Biochemical analysis reveals that CXCR4 and LFA-1 are recruited to lipid rafts upon CD4 but not CD45 engagement. Furthermore, we also demonstrate T cell capping of both lipid rafts and chemokine receptors at sites of contact with HIV-infected cells, despite the binding of an HIV inhibitory mAb to CXCR4. We conclude that cell surface rearrangements in response to CD4 engagement may serve as a means to enhance cell-to-cell signaling at the immunological synapse and modulate chemokine responsiveness, as well as facilitate HIV entry and expansion by synaptic transmission.
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http://dx.doi.org/10.1016/j.yexcr.2004.11.022DOI Listing
April 2005

Quantitative differences in lipid raft components between murine CD4+ and CD8+ T cells.

BMC Immunol 2004 Jan 30;5. Epub 2004 Jan 30.

Laboratory of Immunology, Gerontology Research Center, National Institute on Aging, NIH, Baltimore, MD 21224-6825, USA.

Background: Lipid rafts have been shown to play a role in T cell maturation, activation as well as in the formation of immunological synapses in CD4+ helper and CD8+ cytotoxic T cells. However, the differential expression of lipid raft components between CD4+ and CD8+ T cells is still poorly defined. To examine this question, we analyzed the expression of GM1 in T cells from young and aged mice as well as the expression of the glycosylphosphatidylinositol (GPI)-linked protein Thy-1 and cholesterol in murine CD4+ and CD8+ T cell subpopulations.

Results: We found that CD4+CD8- and CD8+CD4- thymocytes at different stages of maturation display distinct GM1 surface expression. This phenomenon did not change with progressive aging, as these findings were consistent over the lifespan of the mouse. In the periphery, CD8+ T cells express significantly higher levels of GM1 than CD4+ T cells. In addition, we observed that GM1 levels increase over aging on CD8+ T cells but not in CD4+ T cells. We also verified that naïve (CD44lo) and memory (CD44hi) CD8+ T cells as well as naïve and memory CD4+ T cells express similar levels of GM1 on their surface. Furthermore, we found that CD8+ T cells express higher levels of the GPI-anchored cell surface protein Thy-1 associated with lipid raft domains as compared to CD4+ T cells. Finally, we observed higher levels of total cellular cholesterol in CD8+ T cells than CD4+ T cells.

Conclusion: These results demonstrate heterogeneity of lipid raft components between CD4+ and CD8+ T cells in young and aged mice. Such differences in lipid raft composition may contribute to the differential CD4 and CD8 molecule signaling pathways as well as possibly to the effector responses mediated by these T cell subsets following TCR activation.
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http://dx.doi.org/10.1186/1471-2172-5-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC343273PMC
January 2004

Akt forms an intracellular complex with heat shock protein 90 (Hsp90) and Cdc37 and is destabilized by inhibitors of Hsp90 function.

J Biol Chem 2002 Oct 9;277(42):39858-66. Epub 2002 Aug 9.

Program in Pharmacology, Weill Graduate School of Medical Sciences, Cornell University and the Program in Cell Biology and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.

Hsp90 is a chaperone required for the conformational maturation of certain signaling proteins including Raf, cdk4, and steroid receptors. Natural products and synthetic small molecules that bind to the ATP-binding pocket in the amino-terminal domain of Hsp90 inhibit its function and cause the degradation of these client proteins. Inhibition of Hsp90 function in cells causes down-regulation of an Akt kinase-dependent pathway required for D-cyclin expression and retinoblastoma protein-dependent G(1) arrest. Intracellular Akt is associated with Hsp90 and Cdc37 in a complex in which Akt kinase is active and regulated by phosphatidylinositol 3-kinase. Functional Hsp90 is required for the stability of Akt in the complex. Occupancy of the ATP-binding pocket by inhibitors is associated with the ubiquitination of Akt and its targeting to the proteasome, where it is degraded. This results in a shortening of the half-life of Akt from 36 to 12 h and an 80% reduction in its expression. Akt and its activating kinase, PDK1, are the only members of the protein kinase A/protein kinase B/protein kinase C-like kinase family that are affected by Hsp90 inhibitors. Thus, transduction of growth factor signaling via the Akt and Raf pathways requires functional Hsp90 and can be coordinately blocked by its inhibition.
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http://dx.doi.org/10.1074/jbc.M206322200DOI Listing
October 2002